Since the development and the widespread use of chemical insecticides, the occurrence of resistant insect strains has been an important problem. Development of insecticide resistance is a phenomenon dependent on biochemical, physiological, genetic and ecological mechanisms. Currently, insect resistance has been reported against all major classes of chemical insecticides including chlorinated hydrocarbons, organophosphates, carbamates, and pyrethroid compounds (Brattsten et al., 1986).
In contrast to the rapid development of insect resistance to synthetic insecticides, development of insect resistance to bacterial insecticides such as B. thuringiensis sprays has evolved slowly despite many years of use (Brattsten et al., 1986). The spore forming gram-positive bacterium B. thuringiensis produces a parasporal crystal which is composed of crystal proteins (ICPs) having insecticidal activity. Important factors decreasing the probability of emergence of resistant insect strains in the field against B. thuringiensis sprays are: firstly the short half-life of B. thuringiensis sprays after foliar application; secondly the fact that commercial B. thuringiensis preparations often consist of a mixture of several insecticidal factors including spores, ICPs and eventually beta-exotoxins (Shields, 1987); and thirdly the transitory nature of plant-pest interactions. Many successful field trials have shown that commercial preparations of a B. thuringiensis containing its spore-crystal complex, effectively control lepidopterous pests in agriculture and forestry (Krieg and Langenbruch, 1981). B. thuringiensis is at present the most widely used pathogen for microbial control of insect pests.
Various laboratory studies, in which selection against B. thuringiensis was applied over several generations of insects, have confirmed that resistance against B. thuringiensis is seldom obtained. However, it should be emphasized that the laboratory conditions represented rather low selection pressure conditions.
For example, Goldman et al. (1986) have applied selection with B. thuringiensis israelensis toxin over 14 generations of Aedes aegypti and found only a marginal decrease in sensitivity. The lack of any observable trend toward decreasing susceptibility in the selected strains may be a reflection of the low selection pressure (LC.sub.50) carried out over a limited number of generations. However, it should be pointed out that Georghiou et al. (In: Insecticide Resistance in Mosquitoes: Research on new chemicals and techniques for management. In "Mosquito Control Research, Annual Report 1983, University of California.") with Culex quinguefasciatus obtained an 11-fold increase in resistance to B. thuringiensis israelensis after 32 generations at LC.sub.95 selection presssure.
McGaughey (1985) reported that the grain storage pest Plodia interpunctella developed resistance to the spore-crystal complex of B. thuringiensis; after 15 generations of selection with the Indian meal moth, Plodia interpunctella, using a commercial B. thuringiensis HD-1 preparation ("Dipel", Abbott Laboratories, North Chicago, Ill. 60064, USA), a 100-fold decrease in B. thuringiensis sensitivity was reported. Each of the colonies was cultured for several generations on a diet treated with a constant B. thuringiensis dosage which was expected to produce 70-90% larval mortality. Under these high selection presssure conditions, insect resistance to B. thuringiensis increased rapidly. More recently, development of resistance against B. thuringiensis is also reported for the almond moth, Cadra cautella (McGaughey and Beeman, 1988). Resistance was stable when selection was discontinued and was inherited as a recessive trait (McGaughey and Beeman, 1988). The mechanism of insect resistance to B. thuringiensis toxins of Plodia interpunctella and Cadra cautella has not been elucidated.
The main cause of B. thuringiensis resistance development in both reported cases involving grain storage was the environmental conditions prevailing during the grain storage. Under the conditions in both cases, the environment was relatively stable, so B. thuringiensis degradation was slow and permitted successive generations of the pest to breed in the continuous presence of the microbial insecticide. The speed at which Plodia developed resistance to B. thuringiensis in one study suggests that it could do so within one single storage season in the bins of treated grain.
Although insect resistance development against B. thuringiensis has mostly been observed in laboratory and pilot scale studies, very recent indications of B. thuringiensis resistance development in Plutella xylostella populations in the (cabbage) field have been reported (Kirsch and Schmutterer, 1988). A number of factors have led to a continuous exposure of P. xylostella to B. thuringiensis in a relatively small geographic area. This and the short generation cycle of P. xylostella have seemingly led to an enormous selection pressure resulting in decreased susceptibility and increased resistance to B. thuringiensis.
A procedure for expressing a B. thuringiensis ICP gene in plants in order to render the plants insect-resistant (European patent publication ("EP") 0193259 which is incorporated herein by reference!; Vaeck et al., 1987; Barton et al., 1987; Fischhoff et al., 1987) provides an entirely new approach to insect control in agriculture which is at the same time safe, environmentally attractive and cost-effective. An important determinant for the success of this approach will be whether insects will be able to develop resistance to B. thuringiensis ICPs expressed in transgenic plants (Vaeck et al., 1987; Barton et al., 1987; Fischhoff et al., 1987). In contrast with a foliar application, after which B. thuringiensis ICPs are rapidly degraded, the transgenic plants will exert a continuous selection pressure. It is clear from laboratory selection experiments that a continuous selection pressure has led to adaptation to B. thuringiensis and its components in several insect species. In this regard, it should be pointed out that the conditions in the laboratory which resulted in the development of insect-resistance to B. thuringiensis are very similar to the situation with transgenic plants which produce B. thuringiensis ICPs and provide a continuous selection pressure on insect populations feeding on the plants. Mathematical models of selection pressure predict that, if engineered insect-resistant plants become a permanent part of their environment, resistance development in insects will emerge rapidly (Gould, 1988). Thus, the chances for the development of insect resistance to B. thuringiensis in transgenic plants may be considerably increased as compared to the field application of B. thurinqiensis sprays. A Heliothis virescens strain has been reported that is 20 times more resistant to B. thuringiensis HD-1 ICP produced by transgenic Pseudomonas fluorescens and 6 times more resistant to the pure ICP (Stone et al., 1989). Furthermore, the monetary and human costs of resistance are difficult to assess, but loss of pesticide effectiveness invariably entails increased application frequencies and dosages and, finally, more expensive replacement compounds as new pesticides become more difficult to discover and develop.
Therefore, it would be desirable to develop means for delaying or even preventing the evolution of resistance to B. thuringiensis.
B. thuringiensis strains, active against Lepidoptera (Dulmage et al., 1981), Diptera (Goldberg and Margalit, 1977) and Coleoptera (Krieg et al., 1983), have been described. It has become clear that there is a substantial heterogeneity among ICPs from different strains active against Lepidoptera, as well as among ICPs from strains active against Coleoptera (Hofte and Whiteley, 1989). An overview of the different B. thuringiensis ICP genes, that have been characterized, is given in Table 2 (which follows the Examples herein).
Most of the anti-Lepidopteran B. thuringiensis (e.g., Bt3, Bt2, Bt73, Bt14, Bt15, Bt4, Bt18) ICP genes encode 130 to 140 kDa protoxins which dissolve in the alkaline environment of an insect's midgut and are proteolytically activated into an active toxin of 60-65 kDa. These ICPs are related and can be recognized as members of the same family based on sequence homologies. The sequence divergence however is substantial, and the insecticidal spectrum, among the order Lepidoptera, may be substantially different (Hofte et al., 1988).
The P2 toxin gene and the cry B2 gene are different from the above-mentioned genes in that they do not encode high molecular weight protoxins but rather toxins of around 70 kDa (Donovan et al., 1988 and Widner and Whiteley, 1989, respectively).
It has recently become clear that heterogeneity exists also in the anti-Coleopteran toxin gene family. Whereas several previously reported toxin gene sequences from different B. thuringiensis isolates with anti-Coleopteran activity were identical (EP 0149162 and 0202739), the sequences and structure of bt21and bt22 are substantially divergent (European patent application ("EPA") 89400428.2).
While the insecticidal spectra of B. thuringiensis ICPs are different, the major pathway of their toxic action is believed to be common. All B. thuringiensis ICPs, for which the mechanism of action has been studied in any detail, interact with the midgut epithelium of sensitive species and cause lysis of the epithelial cells (Knowles and Ellar, 1986) due to the fact that the permeability characteristics of the brush border membrane and the osmotic balance over this membrane are perturbed. In the pathway of toxic action of B. thuringiensis ICPs, the binding of the toxin to receptor sites on the brush border membrane of these cells is an important feature (Hofmann et al., 1988b). The toxin binding sites in the midgut can be regarded as an ICP-receptor since toxin is bound in a saturable way and with high affinity (Hofmann et al., 1988a).
Although this outline of the mode of action of B. thuringiensis ICPs is generally accepted, it remains a matter of discussion what the essential determinant(s) are for the differences in their insecticidal spectra. Haider et al. (1986) emphasize the importance of specific proteases in the insect midgut. Hofmann et al. (1988b) indicate that receptor binding is a prerequisite for toxic activity and describe that Pieris brassicae has two distinct receptor populations for two toxins. Other authors have suggested that differences in the environment of the midgut (e.g., pH of the midgut) might be crucial.